Enzymatic Ligation of Large Biomolecules to DNA

Abstract

The ability to synthesize, characterize, and manipulate DNA forms the foundation of a range of advanced disciplines including genomics, molecular biology, and biomolecular engineering. In particular for the latter field, DNA has proven useful as a structural or functional component in nanoscale self-assembled structures, antisense therapeutics, microarray diagnostics, and biosensors. Such applications frequently require DNA to be modified and conjugated to other macromolecules, including proteins, polymers, or fatty acids, in order to equip the system with properties required for a particular application. However, conjugation of DNA to large molecular components using classical chemistries often suffers from suboptimal yields. Here, we report the use of terminal deoxynucleotidyl transferase (TdT) for direct enzymatic ligation of native DNA to nucleotide triphosphates coupled to proteins and other large macromolecules. We demonstrate facile synthesis routes for a range of NTP-activated macromolecules and subsequent ligation to the 3' hydroxyl group of oligodeoxynucleotides using TdT. The reaction is highly specific and proceeds rapidly and essentially to completion at micromolar concentrations. As a proof of principle, parallelly labeled oligonucleotides were used to produce nanopatterned DNA origami structures, demonstrating rapid and versatile incorporation of non-DNA components into DNA nanoarchitectures.

DNA nanotubes as intracellular delivery vehicles in vivo

“…To visualize the DNA nanotubes in vivo, the 3 0 ends of some of the tile strands were enzymatically labeled with Atto488-dUTP or Cy3-dUTP [31]. For this, Atto488-dUTP or Cy3-dUTP (80 mM, purchased from Jena Bioscience, Jena, Germany), CoCl 2 (5 mM), terminal transferase enzyme (16 U/ml, Roche, Penzberg, Germany), and all DNA tiles (400 pmol) were mixed in a 20 ml, 1Â TdT reaction buffer.…”

DNA Nanostructures as Programmable Biomolecular Scaffolds

“…71 There is another option for replacing DNA modification by using terminal deoxynucleotidyl transferase (TdT), which can ligate native DNA to proteins coupled with nucleotide triphosphates. 72 TdT can accept nucleoside triphosphates tethered to large biomolecules as substrates and direct the ligation of the biomolecules to the 3′ end of any native oligodeoxynucleotides (Scheme 5C). The reaction is rapid and quantitative, while in mild and aqueous conditions.…”

Sequenz‐spezifischer Einbau von Enzym‐Nukleotid‐Chimären durch DNA‐Polymerasen

“…[18] Darüber hinaus wurden effiziente Methoden für die Verknüpfung von modifizierten Nukleotiden und Proteinen nach dem Einbau durch die DNA-Polymerase entwickelt. [18] Darüber hinaus wurden effiziente Methoden für die Verknüpfung von modifizierten Nukleotiden und Proteinen nach dem Einbau durch die DNA-Polymerase entwickelt.…”